Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K_r range from approximately 2 x 10^4 to approximately 2 x 10^6 for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^5 to 2 x 10^6 molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ⩽10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage

Intersecting batteries of differentially expressed genes in the early sea urchin embryo

We determined the distribution of cis-regulatory sites, previously identified in the control domain of the CyIIIa gene, in three other genes displaying diverse spatial patterns of expression in the sea urchin embryo. Competitive gel-shift reactions were carried out using probes from the CyIIIa gene, with competitor fragments isolated from the previously defined control domains of the other genes. CyIIIa is expressed only in aboral ectoderm lineages; the other genes studied were Spec1, also expressed in aboral ectoderm; CyI, expressed in many different cell types; and SM50, expressed only in skeletogenic mesenchyme. All four genes are activated at about the same time in late cleavage. Where competitive interactions indicated a functionally comparable binding site (in vitro), a sequence homology was sought, and in most cases could be identified. An interesting pattern of putative regulatory site usage emerges: Of 10 CyIIIa interactions tested, three only were unique to the CyIIIa gene with respect to the set of four genes tested; one believed on previous evidence to be a temporal regulator was shared by all four genes, and the remainder were shared in various subsets of the four genes

Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K_r range from approximately 2 x 10^4 to approximately 2 x 10^6 for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^5 to 2 x 10^6 molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ⩽10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage

Synthesis and characterization of new polyesters based on renewable resources

A series of non-crosslinked biobased polyesters were prepared from pentaerythritol and aliphatic dicarboxylic acids, including fatty acids grafted as side-chains to the backbone of the polymer. The strategy utilized tends to create linear polymers by protecting two of the hydroxyl groups in pentaerythritol by esterification with fatty acids before the polymerization reaction. The solvent-free syntheses were performed under vacuum and catalyzed by the ion-exchange resin Amberlyst 70. The maximum yield was around 98%. Pristine polyesters had average molecular weights of about 104 g/mol according to SEC-MALLS analysis. Melting temperatures and extent of crystallinity were determined by differential scanning calorimetry. By using relatively short fatty acids, such as lauric acid, soft materials were obtained with low crystallinity and a melting point below room temperature, whereas longer side-chains, such as behenic acid, gave brittle polymers with higher melting temperatures and crystallinity. The use of a short chain dicarboxylic acid, such as succinic acid, resulted in closer side-chains and promoted higher crystallinity and melting temperatures. In order to improve the thermal properties of these materials, a series of copolyesters were designed by developing synthetic methods to approach a random- and a block-copolymerization. A wide range of properties was thus obtained according to the composition of these novel copolyesters

Synthesis of new cellulose ethers using Suzuki–Miyaura reactions

Cellulose ethers are functionalized biopolymers that are industrially produced through drastic conditions employing gaseous reactants with a high risk of industrial accident. The cellulose ethers that are commercially available generally bear short carbon-chains. In this work, an alternative method using non-gaseous chemicals is proposed. It relies on the use of the Suzuki–Miyaura reaction employing mild, moisture- and air-stable conditions. Relatively innocuous reagents are used for this step, which allows the formation of a wide range of cellulose ethers bearing various functional groups with different chain-length

Management of upper gastrointestinal bleeding in emergency departments, from bleeding symptoms to diagnosis: a prospective, multicenter, observational study.

BACKGROUND: Upper gastrointestinal bleeding (UGB) is common in emergency departments (EDs) and can be caused by many eso-gastro-duodenal lesions. Most available epidemiological data and data on the management of UGB comes from specialized departments (intensive care units or gastroenterology departments), but little is known from the ED perspective. We aimed to determine the distribution of symptoms revealing UGB in EDs and the hemorrhagic lesions identified by endoscopy. We also describe the characteristics of patients consulting for UGB, UGB management in the ED and patients outcomes. METHOD: This was a prospective, observational, multicenter study covering 4 consecutive days in November 2013. Participating EDs were part of the Initiatives de Recherche aux Urgences network coordinated by the French Society of Emergency Medicine. All patients with suspected UGB in these EDs were included. RESULTS: In total, 110 EDs participated, including 194 patients with suspected UGB (median age 66 years [Q1-Q3: 51-81]). Overall, 104 patients (54%) had hematemesis and 75 (39%) melena. Endoscopy revealed lesions in 121 patients, mainly gastroduodenal ulcer or ulcerations (41%) or bleeding lesions due to portal hypertension (20%). The final diagnosis of UGB was reversed by endoscopy in only 3% of cases. Overall, 67 patients (35%) had at least one severity sign. Twenty-one patients died (11%); 40 (21%) were hospitalized in intensive care units and 126 (65%) in medicine departments; 28 (14%) were outpatients. Mortality was higher among patients with clinical and biological severity signs. CONCLUSION: Most of the UGB cases in EDs are revealed by hematemesis. The emergency physician diagnosis of UGB is rarely challenged by the endoscopic findings

Etude de la différenciation musculaire lisse chez Xenopus Laevis (induction et régulation dans un modèle de cellules embryonnaires pluripotentes)

Notre travail a porté sur la différenciation musculaire lisse dans le modèle amphibien Xenopus laevis. Nous avons cloné et caractérisé les ADNc codant pour les protéines spécifiques de la cellule musculaire lisse myosine (SM-MHC), l'a-actine lisse et SM22a. Ces outils nous ont permis d'établir que l'induction du programme myogénique musculaire dans l'embryon était prédéterminé bien avant l'expression des marqueurs structuraux. Nous avons développé un modèle de différenciation musculaire lisse à partir d'explants de cellules ectodermiques prélevés sur des embryons de stade blastula. Ces explants peuvent se différencier en CML lorsqu'ils sont traités par le facteur de croissance bFGF et cette différenciation peut être modulée par différentes molécules de signalisation comme Wnt8, BMP2 ou BMP4. Seuls certains gènes musculaires lisses peuvent être induits dans ces explants par la surexpression de différents facteurs de transcription GATA, Nkx2.5, HAND, MEF2 ou SRF. Le modèle de cellules embryonnaires que nous avons caractérisé semble particulièrement adapté à l'étude des premières étapes de la différenciation musculaire lisse.Our work has focused on the smooth muscle cell differenciation in the Xenopus laevis amphibian model. We have cloned and caracterized cDNAs encoding the smooth muscle cell specific proteins myosin heavy chain, smooth muscle a-actin and SM22a. These clones allowed us to show that the induction of the smooth muscle myogenic programm is established early in the embryo well before the detectable expression of structural markers. We have developped a model of smooth muscle differentiation based on animal cap explants from blastula stage embryos. These explants can differentiate along the smooth muscle pathway when they are treated with appropriate concentrations of the growth factor bFGF. This differentiation can be modulated by different signaling molecules like Wnt8, BMP2 ou BMP4. Some of the smooth muscle genes studied can be induced in these explants after oveexpression of transcription factors like GATA, Nkx2.5, HAND, MEF2 or SRF. The model of pluripotent embryonic cells we have developped is suitable for the study of the first steps of smooth muscle cell differentiation.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Etude chez le xénope (de la différenciation musculaire lisse et de la fonction des gènes C3H au cours du développement embryonnaire)

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF